We propose to use two methods involving specific labeling of proteins to obtain information concerning enzyme conformation and mechanism. Enzymes specifically labeled with C-13 or F-19 nuclei will be obtained by incorporation of labeled amino acids or by chemical modification of intact enzymes with labeled reagents. The labeled enzymes and their noncovalent complexes with substrates, inhibitors, and coenzymes, will be studied by means of nuclear magnetic resonance. The active site of tryptophan synthetase A protein, the NAD binding site of horse liver alcohol dehydrogenase, the CTP binding site of aspartate transcarbamylase, and active sites of several other dehydrogenases will be studied by this technique. The wide chemical shift ranges of C-13 and F-19 nuclei and the relative narrowness of their nmr signals should make C-13 and F-19 nmr extremely valuable in determining the structure of macromolecules in solution. The second approach to be employed involves photoaffinity labeling of the pyrimidine binding sites of ribonuclease A and staphylococcal nuclease. The identity of the amino acid residues modified when the noncovalent complexes of these enzymes and pyrimidine nucleotides are irradiated will be determined. This information will allow the structure of the binding sites to be inferred.